The long-term goal of this proposal is to define molecular mechanisms that regulate the trafficking of integral membrane proteins to the lysosome for degradation. The ESCRT machinery, a set of conserved endosomal protein complexes, is proposed to bind directly to ubiquitinylated membrane proteins and govern their entry into vesicles that bud into the lumen of specialized multivesicular endosomes (MVEs). This process is particularly important for the downregulation of hormone receptors and to prevent constitutive signaling, which can lead to developmental abnormalities and disease. How the late-acting components of the ESCRT machinery coordinate the formation of intraluminal vesicles at MVEs will be addressed in this proposal. The C. elegans germline and early embryo are powerful model systems to study membrane dynamics in an intact, developing animal. Specific proteins can be efficiently depleted from oocytes using RNA interference. Additionally, oocyte maturation and fertilization reproducibly trigger the internalization and ESCRT-mediated degradation of multiple transmembrane proteins, providing an ideal, physiologically relevant system for studying lysosomal protein transport. C. elegans is highly amenable to genetic manipulation and can be engineered easily to stably express transgenes for gene replacement strategies. Additionally, we have established methods to high pressure freeze animals at specific time points during embryo development to enable the stepwise characterization of de novo MVE biogenesis using electron microscopy (EM)-based approaches. Given the stereotypic nature of early embryo development, we can correlate these EM data directly with our findings using live cell imaging assays, which we have pioneered in this system. Taking advantage of this unique combination of attributes, the specific aims of this first renewal application are to: ) define regulatory mechanisms that specify the site of ILV formation on MVEs, 2) determine mechanisms that promote the nucleation of ESCRT-III filaments, and 3) define regulatory mechanisms that control ESCRT-III polymer dynamics. The genetic and biochemical studies conducted during the first period of grant support defined new methods and tools to study ESCRT-III polymer assembly, raising intriguing hypotheses regarding how this process is controlled. Using a combination of in silico molecular modeling, in vitro reconstitution experiments, and in vivo high resolution microscopy-based assays, we will define new mechanisms that regulate ESCRT-III complex assembly during MVE formation. These studies will provide a key framework for future investigation into highly related pathways in mammalian cells.